(a) Field of the Invention
The present invention relates to a composition for detecting peptidoglycan, and a diagnostic kit for peptidoglycan using the same.
(b) Description of the Related Art
The infection of the pathogenic Gram-positive bacterium constitutes a large portion of widely-spread bacterial infection in hospitals. Food poisonings and bacterial sepsis caused by Gram-positive bacterium are lethal diseases. A rapid detecting system for Gram-positive bacteria in clinical samples such as blood, tissue, and urine, and food is necessary.
According to a conventional technique, it takes a few days to detect the bacteria. As foods contaminated with the bacteria will be distributed during this detection period, additional consumers may be infected.
It is possible to detect Gram-positive bacteria living in various types of samples in small amounts by detecting and quantifying the peptidoglycan.
The peptidoglycan is a kind of glycoprotein polymer constituting bacterial cell walls and contains N-acetylmuramic acid or N-glycosylmuramic acid and D-amino acid in outer cell wall of Gram-positive bacteria.
Accordingly, a detecting and measuring method of the peptidoglycan can be applied for testing the safeness of the medicine, detecting microorganism in food and water, and performing diagnosis of infectious disease.
It is reported that a prophenoloxydase system of insect can detect selectively a small amount of lipopolysaccharide (LPS), peptidoglycan, and beta-1,3-glucan, in which zymogen-type prophenoloxydase is converted to phenoloxidase in active form through cascade reactions to amplify the signal more than 1,000 times, However, the prophenoloxydase system detects all components including lipopolysaccharide, peptidoglycan, and beta-1,3-glucan. Therefore, a system for selectively detecting any one of them is necessary.
A prophenoloxidase, which exists in insect body with complete metamorphosis, is activated to phenoloxidase through cascade reaction on the beta-1,3-glucan or lipopolysaccharide. The prophenoloxidase reaction system consisting of a series of cascade reaction steps can be easily activated to phenoloxidase system to produce melanin by using catechol amines when it is exposed to exterior factors such as a pathogenic microorganism and materials, or interior factors derived from degranulation of interior hemocyte, etc. Thus, it is difficult to extract the prophenoloxidase system from the insect body (Ashida and Yoshida, (1988), Insect. Biochem. 18, 11-19).
Ashida et al. reported in Eur. J. Biochem, 188, 507-515 (1990) that bivalent ion plays an important role in activating prophenoloxidase system by introducing a composition which recognizes beta-1,3-glucan separated from mosquito larva, and disclosing that prophenoloxidase system of insects requires Ca2+ for its activation.
A composition and method for detecting peptidoglycan was disclosed in U.S. Pat. No. 4,970,152 where a protein reacting with beta-1,3-glucan was removed from silkworm plasma to produce a reagent for specifically detecting a peptidoglycan. However, the addition of Ca2+ or Mg2+ is required for activating a phenoloxidase system on peptidoglycan. In other words, according to U.S. Pat. No. 4,970,152, inhibition of the phenoloxidase system activation by adding Ca2+ is necessary when obtaining a phenoloxidase composition from the insect body fluid and triggering a color reaction on the peptidoglycan as a substrate with the composition.
Also, U.S. Pat. No. 5,747,277 disclosed a SLP reagent. However, this reagent detected beta-1,3-glucan and peptidoglycan at the same time; therefore, it did not show a specific reaction to only peptidoglycan. As result, this reagent cannot be used for the detection of only peptidoglycan. Additionally, the SLP reagent includes Ca2+.